Regeneration of mammalian tissues using synthetic immodulins

ABSTRACT

Methods and compositions involving synthetic immodulin peptides and helper molecules. The peptides exhibit new and surprising biological activities, such as co-stimulation of macrophage and myogenic cell differentiation markers. Methods are provided N by which peptide is contacted with one or more myeloid precursor cell populations, thereby increasing the abundance of CD169+, CCL22+, Clec4A, Clec9a+, and Clec 12a+ monocyte lineages which play important roles in cross presentation, post-apoptotic clearance, autoimmunity, and programmatic tissue regeneration, notably in contexts of tissue stress, insult and degeneration. The disclosed methods and compositions enable concurrent regeneration of diverse cellular elements in tissue where collaborating myeloid and non-myeloid lineages are located together in a living tissue following the contacting steps. Furthermore, the peptides of the invention can be delivered with inherently specific in vivo targeting, achieved through complexation to holotransferrin and/or size-specific glycosaminoglycans (e.g. high-molecular- weight hyaluronan) so as to discriminate between target environments in vivo or ex vivo.

This application claims benefit of international application number PCT/US2021/046814, with an international filing date of 20 Aug. 2021, and international application number PCT/US2021/021433, with an international filing date of 9 Mar. 2021. PCT/US2021/046814 claims priority to international application number PCT/US2021/021433, which claims priority to international application number PCT/US2020/024828 with an international filing date of 26 Mar. 2020.

TECHNICAL FIELD

This invention relates to the field of peptide therapeutics, and more particularly to the use of synthetic “immodulin” peptides (also optionally referred to as “immodulator peptides”) to selectively increase the abundance of two or more cooperating mammalian cell lineages in vivo or ex vivo. Cooperating lineages may include cells involved in the arbitration of inflammation (e.g. macrophages) followed, programmatically, by the regeneration of new cells in the same tissue. The compositions and methods provided herein demonstrate new and surprising uses of immodulin peptides for the regeneration of tissues in mammals.

SUMMARY OF THE INVENTION

This invention provides synthetic immodulin peptides, originally derived from the sequences of classical insulin growth factor binding proteins (IGFBPs 1 through 6), and related compositions and methods. Insulin-like growth factors (IGFs) influence the growth and differentiation of mammalian cells. Unlike the full-length classical IGFBPs, whose primary function is to carry and deliver IGFs, the peptides of this invention are derived from less than 10% of the IGFBP amino acid sequence(s) and do not themselves bind IGFs. Thus, the effects of the synthetic immodulin peptides of this invention are unrelated to the effects of IGFs themselves. IGF-independent effects of classical IGFBPs have been known for decades, but there is no report of (IGF-independent) effects of IGFBP-derived peptides on monocyte differentiation, for example. Furthermore, tissue regeneration requires the coordinated action of multiple cell types. For example, certain myeloid lineages (notably macrophages specialized for anti-inflammatory and pro-efferocytotic functions) interact with tissue-resident precursor lineages for differentiation into roles vacated by cells that have recently succumbed to insult, such as ischemic, oxidative or inflammatory insult. It is a fundamental and central need in regenerative medicine to provide interventions that restore homeostasis to damaged, dysfunctional or degenerated tissue—such as by processing apoptotic cells efficiently through efferocytosis, restoration of mitochondrial integrity and resolution of inflammation by enhancing tolerization—while also providing appropriate stimulus for the differentiation of resident (and sometimes non-resident) precursor cells to take the place of departed cells. A holy grail in the field of regenerative medicine is the discovery of master regulatory molecules capable of conducting this type of programmatic cellular symphony. The synthetic immodulin peptides disclosed herein exhibit new and surprising biological effects on cellular differentiation, such as the stimulation of differentiated myeloid lineages that coordinate the disposal, re-programming and de novo differentiation of adult non-myeloid lineages from precursor cell populations in the tissue. For example, compositions and methods are disclosed herein for increasing the abundance of CD169+, CCL22+, and Clec12a+ cross-presenting myeloid lineages by contacting precursor cells in vivo or ex vivo with synthetic immodulin peptides, while also independently stimulating other, non-myeloid cell types in the tissue, such as myoblasts, to differentiate into complementary roles consistent with tissue regeneration and homeostasis. Such complementary myeloid and non-myeloid cell differentiation activities have never been disclosed for IGFBPs or for IGFBP-derived synthetic peptides. For example, in the over 7000 scientific papers published on IGFBP-3 and IGFBP-3-derived peptides in the past forty years, there is no disclosure of stimulation of differentiation of a specific monocytic or myogenic lineage using synthetic peptides derived from IGFBP-3 (or other classical IGFBP).

The present invention describes the use of synthetic immodulin peptides to increase the abundance of mammalian cell lineages within a living animal or in a population of mammalian cells derived from an animal (i.e. in vivo or ex vivo). Importantly, the action of these peptides is IGF-independent, as these peptides do not bind IGFs. Cells treated with synthetic immodulin peptides ex vivo may subsequently be re-introduced into a living mammal. For example, hematopoietic or other mesenchymal cell lineages can be expanded so as to improve outcomes in cancer therapy, targeted vaccination, management of traumatic insults, anemia, neurodegeneration, regeneration of tissues, and prevention of medical complications from diseases such as obesity, diabetes, and diseases of aging.

Synthetic immodulin peptides are believed to rapidly target and enter cells under stress, move to specific cellular compartments (e.g. the nucleus, cytoplasm, mitochondria), and interact with cellular machinery in different ways. For example, they can bind transcriptional factors and thereby alter large transcriptional sets.

The use of short classical IGFBP-derived synthetic sequences to increase the abundance of differentiation markers in myeloid hematopoietic cell lineages (such as CD169+, CCL22+ or Clec12a+ monocytes, dendritic cells and macrophages) or in non-myeloid lineages important in re-establishment of tissue homeostasis (such as myoblasts, osteoblasts, chondroblasts, neuroblasts, fibroblasts and lymphocytes has never been disclosed. Indeed, to the inventor's knowledge, no other peptide molecule has ever been identified that is capable of such function. As tools for accomplishing targeted differentiation of mammalian cell precursors in vivo or ex vivo, the peptides of this invention are uniquely useful, for example, in the treatment of acute (driven by traumatic insult or myocardial infarction, for example) and chronic inflammatory skeletal myopathies, which have themselves been shown to trigger neurodegeneration, kidney disease, heart failure, COPD, and many of the degenerative conditions associated with aging. Numerous other applications secondary to reversing inflammatory loss of skeletal muscle will be readily apparent to skilled practitioners in the field.

Retinoid X receptors (RXRα (NR2B1), RXRβ (NR2B2) and RXRγ (NR2B3)) are promiscuous heterodimeric partners for other members of the Nuclear Receptor (NR) superfamily including members of the NR1 group, which include thyroid receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator-activated receptors (PPARs), LXRs, FXR and many others; NR3 group (ERs, GR, MR, PR, AR); NR4 (notably NR4A1/Nur77); and members of other NR groups. RXRs are obligatory partners for a number of NR partners, placing RXRs in a coordinating role at the crossroads of multiple signaling pathways. RXRs represent important targets for pharmacologic interventions and therapeutic applications. RXRs function as master regulators producing diverse physiological effects through the activation of multiple nuclear receptor complexes. Immodulin peptides bind RXRs and Nur77 in vitro, and appear to guide heterodimer formation. RXR agonists alone, partner receptor agonists alone or a combination of both can activate dimers, including permissive heterodimers. Such complexes include those formed with PPARs such as PPARα, PPARβ PPARd PPARγ), FXR (farnesoid X receptor), LXR (liver X receptor), and the orphan NR4A group, including Nur77 and Nurr1. Nur77 and Nurr1 transcriptional activities can be indirectly manipulated through modulation of their heterodimeric partner RXR, using ligands such SR11237, BRF110, HX531 or HX600, or other RXR/NR4A ligands and modulators such as spironolactone, haloperidol, cytosporone B, C-DIM12, C-DIM8 and cilostazol; PPAR ligands such as fenofibrate, fenofibric acid, ciprofibrate, gemfibrozil, clofibric acid, elafibrinor, GW9578, RB394, MBX-8025, GW7647, ZLY032, GW590735, GW0742, GW501516 and Amorfrutin B; and NR ligands such as BMS195614, GW4064, BMS453, sobetirome, ciloflexor, TTNPB, adapalene and GW3965.

Classical IGFBPs, from which the core sequences of the immodulin peptides of this invention are derived, are a highly conserved family of proteins, both structurally and functionally. The sequence of IGFBPs 3, 5 and 6 from which the peptides of this invention are derived are particularly closely conserved. These three IGFBPs form a major evolutionary Glade that diverged from IGFBPs 1, 2 and 4 more than 700 million years ago. Functional features present in the 3/5/6 Glade and not in the 1/2/4 Glade include metal binding, PIP2-binding and nuclear transport. Amino acid sequence identity in the C-terminal thyroglobulin type-1 domain from which the sequences of the immodulin peptides of this invention are derived, which is present in all classical IGFBPs, is higher within the 3/5/6 IGFBP Glade (for example, 59% between IGFBP-3 and -5, but only 31% between IGFBP-1 and -5). No synthetic immodulin peptide binds IGFs. Each immodulin sequence represents less than 10% of the original IGFBP sequence from which it derives. It would be surprising if an immodulin retained any of the biological activities of the IGFBP molecule, let alone allow prediction of which. The USPTO has explicitly acknowledged this fact by issuing numerous patent claims drawn to immodulin peptide sequences, even if the biological activities exemplified in those patents were also seen with the parent molecule, for example, metal-binding, or nuclear transport. (See U.S. Pat. Nos. 5,519,003/5,783,405/6,165,977/6,262,023/6,342,368/6,423,684/6,855,693/6,933,275/7,393,835/8,536,135/10,369,191). However, in this case, the inventor is not even aware of any prior disclosure wherein IGFBP proteins alone were successfully used to drive differentiation of mammalian monocyte, myogenic, osteogenic and lymphocytic lineages, and knows of no other peptide in biology that can do so. This is the first demonstration that short synthetic peptides containing less than 10% of an IGFBP sequence can trigger mammalian cell differentiation, notably in multiple cell types of critical importance to establishing tissue regeneration and homeostasis. The immodulin peptide -3, -5 and -6 core sequences of this invention are highly related and comprise, respectively:

SEQ ID NO: 1 GFYKKKQCRPSKGRKRGFCW SEQ ID NO: 2 GFYKRKQCKPSRGRKRGICW SEQ ID NO: 3 GFYRKRQCRSSQGORRGPCW

Successful use of synthetic peptides in medicine faces several practical obstacles relating to manufacture, targeted delivery and potency. The inventor has shown that selective use of D-amino acids at the C-terminal end of immodulin peptides helps both stability and potency. Furthermore, synthetic immodulin peptides of this invention are made additionally potent by covalent attachment to small molecules. Of dozens of small molecules tested, less than half gave industrially useful yields under the harsh conditions of conventional peptide synthesis. This is a surprising and unforeseen result. Moreover, the small molecules successfully used and disclosed herein had not, in most cases, been previously reported as adducts to other peptides. Thus, success in the creation of this new class of chemically modified peptides under industrially expedient manufacturing conditions is strictly trial-based.

Novel features of immodulin peptides that aid in targeted delivery in a real-world context have significant implications in the translational success of this class of molecules in the marketplace. Immodulin peptides naturally bind iron and holotransferrin, a major iron-carrier in the circulation. This invention provides holotransferrin-bound immodulin peptide capable of discriminating phosphatidylinositol 4,5-bisphosphate (PIP2)-rich membrane domains. Immodulins 3/5/6 bind PIP2, which competes with transferrin for binding to peptides. Holotransferrin-bound immodulin 3 or 5 selectively binds high-molecular weight hyaluronan (HMW-HA) more strongly than low-molecular weight hyaluronan (LMW-HA), a key discriminant in selective delivery in vivo, as LMW-HA has been widely associated with inflammatory effects, whereas HMW-HA has been associated with anti-inflammatory effects of hyaluronan. Hyaluronan is an abundant glycosaminoglycan ubiquitously present in the extracellular matrix of cell targets. Even more surprisingly, low endocytic pH dramatically increases binding of holotransferrin-immodulin complex to HMW-HA, but not LMW-HA. Furthermore, the master receptor, CD44, a hyaluronan receptor associated with cellular architecture and differentiation, binds this holotransferrin-immodulin-HMW-HA trimeric complex in vitro. Coupled with the established and ubiquitous role of CD44 and of a recently described endocytic nuclear iron-glycan uptake pathway that is CD44-associated, the intrinsic nuclear translocation and iron-binding properties of immodulins may provide a mechanistic link to the profound influences exerted by immodulins in the context of tissue regeneration. The synthetic peptides of the present invention thus benefit from selective carriage to target cells (such as mesenchymal cells under stress, as shown in references provided), selective uptake by receptors located in favorable membrane domains (such as PIP2-rich domains), selective transfer to a glycan carrier under the low pH conditions of an endocytic environment (such as HMW-HA and HMW-HA::CD44 complex), efficient nuclear import (using the nuclear import sequence contained in the immodulin peptides, as detailed in th references), and the ability to access chromatin wherein RXRs can control transcriptional programs (by stimulation of binding in heterodimeric complexes with NR partners, or by modification of kinase or HDAC activity, for example, as shown in priority documents and in this document).

This invention discloses new compositions and new or improved utilities for the immodulin peptide class. Sequence extensions to immodulin core sequences disclosed in the invention confer new biological activities useful in treating human disease and in cosmetics. Previously known utilities of immodulin peptides are described, for example, in PCT/US2020/024828, PCT/US2021/021433, PCT/US2021/046814, U.S. Pat. Nos. 5,519,003/5,783,405/6,165,977/6,262,023/6,342,368/6,423,684/6,855,693/6,933,275/7,393,835/8,536,135/10,369,1919; and references cited therein. These are all hereby incorporated by reference.

Methods disclosed in this invention include the administration of pharmaceutical compositions containing immodulin peptides to a mammal showing symptoms that may be linked to disease conditions, including but not limited to metabolic, muscle-wasting, neurodegenerative and cardiovascular diseases (especially those characterized by some underlying combination of insulin resistance, hyperglycemia, hypertension or hyperlipidemia); immune response to targeted vaccination, cancer progression and metastasis, pulmonary distress and acute kidney injury (AKI) in critical care settings, sepsis, anemia, systemic inflammatory conditions such as shock, post-operative oxidative stress such as after cardiopulmonary bypass or transplant, burns, blunt trauma, pancreatitis, rhabdomyolysis, xenobiotic stresses caused by cocaine, nicotine, alcohol, aminoglycoside antibiotics, cyclosporins, antiviral compounds or chemotherapeutic agents such as platinum compounds or doxorubicin; neuropathic pain and migraine; neurodegenerative diseases such as major depression, Parkinson's, Alzheimer's, Huntington's and ALS/Lou Gehrig's disease; immunosuppression phenomena; chronic obstructive pulmonary disease and other pulmonary diseases; pathological angiogenesis; impaired wound healing; ototoxicities; autoimmune conditions such as lupus erythematosus, arthritis, psoriasis, colitis, fibromyalgia, and multiple sclerosis; genetic diseases such as immune insufficiencies; cystinosis, Fanconi's and other conditions affecting mitochondrial respiration; other forms of mitochondrial dysfunction of bioenergetic failure; pulmonary diseases, especially chronic obstructive pulmonary disease, pulmonary arterial hypertension and asthma; ocular diseases such as cataracts and retinopathies, and conditions caused by infectious agents, including chronic viral infections such as hepatitis, influenza and coronavirus.

In one aspect, the invention provides a synthetic peptide, 20-60 amino acids in length, comprising: (i) a core immodulin sequence corresponding to any one of SEQ ID NOs:1-6; and, optionally, (ii) a small molecule of molecular mass less than one thousand daltons linked covalently to the amino terminus of the peptide. In some embodiments the small molecule is selected from the group consisting of: oleic acid, lauric acid, 2-hydroxy-2-decenoic acid, phenolic acids, anthraquinones, pentacyclic triterpenoids, retinoic acids, bexarotene and other rexinoids, rhein, proprionic acids, TLR4 inhibitors, keto acids, cinnamic acids, aromatic carboxylic acids, indoleacetic acids, xanthenes, xanthones, 2,7-dichlorodihydro-fluorescein diacetate, indolyl-carboxylic acids, PF-06409577, AICAR, D942, PT1, EX-229, GIT27, GW501516, GW3965, GW9578, RB394, MBX-8025, GW7647, ZLY032, GW590735, GW0742, Amorfrutin B, BMS195614, GW4064, BMS453, FTY720, artesunate, artemisinic acid, sobetirone, cilofexor, decanoic acid, eicosapentaenoic acid, docosahexanoic acid, lignoceric acid, TTNPB, adapalene, bexarotene, transcinnamic acid, fenofibric acid, ciprofibrate, chlorfibric acid, gemfibrozil, elafibrinor, pioglitazone, roziglitazone, valproic acid, 2-hexyl-4-pentynoic acid, ibuprofen, C646, SR11237, MSA-2, SR-717 and bromopyruvic acid.

In some embodiments a synthetic immodulin peptide is complexed or co-administered with a metal selected from the group consisting of: ferrous iron, ferric iron, zinc, copper, ruthenium, cobalt, titanium and calcium.

In some embodiments a synthetic immodulin peptide described herein is complexed or co-administered with a glycosaminoglycan or other extracellular matrix component including a group consisting of: collagen, transferrins, other iron-binding proteins, heparin, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, and hyaluronate.

In some embodiments a synthetic immodulin peptide described herein is co-administered with a helper molecule selected from a group consisting of: phytochemicals, ornithine, leucine, raffinose sugar, trehalose sugar, avocado sugar, lactate, bile salts, cyclodextrin, resveratrol, polydatin, ursolic acid and cyclic dinucleotides.

In a related aspect, the invention provides a pharmaceutical composition that includes any synthetic immodulin peptide or peptide complex described herein, and a pharmaceutically acceptable excipient.

In yet another related aspect the invention provides a method for treating a subject suffering from immunological, neurological, myopathic, oncologic, skeletal, reproductive, metabolic or cosmetic dysfunction or imbalance, where the method includes administering to the subject (e.g., a human subject) a therapeutically effective dose of a synthetic immodulin peptide or immodulin peptide/helper molecule complex, or a pharmaceutical composition as described herein. In some embodiments the therapeutically effective dose of the synthetic immodulin peptide is from about 0.01 mg/kg/day to about 50 mg/kg/day.

In yet another related aspect, the invention provides an in vitro method for using cultured mammalian cells to measure the potency of any immodulin peptide described herein.

The compositions of the invention may be administered by means that include but are not limited to intravenous, oral, subcutaneous, intraarterial, intramuscular, intracardial, intraspinal, intrathoracic, intraperitoneal, sublingual, transdermal, intranasal and by inhalation.

DETAILED DESCRIPTION

The terms “subject” and “individual”, as used herein, refer to mammalian individuals, and more particularly to pet animals (e.g., dogs, cats), agricultural animals (e.g., cows, horses, sheep, and the like), and primates (e.g., humans).

The term “treatment” is used herein as equivalent to the term “alleviating”, which, as used herein, refers to an improvement, lessening, stabilization, or diminution of a symptom of a disease or immune imbalance. “Alleviating” also includes slowing or halting progression of a symptom.

As used herein, “co-administration”, “in conjunction with”, “concurrent”, or “concurrently”, as used interchangeably herein, refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality just before, during or soon after delivery of the other treatment modality to the subject.

The term “synthetic immodulin peptide” shall mean a peptide molecule 20-60 amino acids in length prepared by chemical synthesis and comprising any of SEQ ID NOs:1-6.

The term “phytochemical” shall include D-heptomannulose, trehalose, naringin, resveratrol, polydatin, plumbagin, quercetin, curcumin, berberine, alpha-mangostin, wogonin, and ursolic acid.

The term “rexinoid” includes all ligands of RXRs, and conjugates thereof.

The term “bexarotene-class rexinoid” shall include bexarotene, LG100268, SR11237, HX600, HX531, BRF110 and conjugates thereof.

The term NSAID includes ibuprofen, sulindac (and its sulfide and sulfone derivatives), indomethacin, aspirin, naproxin, ketoprofen, ketorolac, diclofenac and etodolac, and conjugates thereof.

The term “RLR/STING/IFN-class agonist” shall include cyclic dinucleotides such as 2′3′cGAMP and cyclic di-GMP, nucleotides such poly-I:C and double-stranded ppp-RNA, and small molecule agonists such as G10, KIN1400, KIN1408, KIN 1148, R08191, MSA-2, SR-717, alpha-mangostin, DMXAA and conjugates thereof.

The term “NR4A-class ligand” shall include spironolactone, haloperidol, cytosporone B, C-DIMS, C-DIM8, C-DIM12, cilostazol, PDNPA and conjugates thereof.

The term “immunomodulant-class molecule” shall include GIT-27, Schisandrin A, resiquimod (R-848), hydroxychloroquine, pidotimod, itraconazole, homoharringtonine, salidroside, celastrol, zymostenol, 7-dehydrocholesterol and conjugates thereof.

The term “Wnt-class molecule” shall include gallic acid, methyl gallate, gallocyanine, epigallocatechin gallate, XAV939, ethacrynic acid, leonurine and conjugates thereof.

“Significantly increasing the abundance of a mammalian cell lineage differentiation marker” shall mean increasing the relative average abundance of the cell lineage differentiation marker in the population of cells contacted with synthetic immodulin peptide by at least fifteen percent over an untreated population control, such that the average difference between the two populations is statistically significant—for example, with a calculated probability of p<0.05 using Student's T-test, or other comparable statistical test, well known to those skilled in the art. “Differentiated mammalian cell lineage” means a living mammalian cell population expressing markers and/or functions characteristic of a differentiated cell type. “In vivo” treatment means treatment within a living animal. “Ex vivo” treatment means treatment of a mammalian cell population after its removal from an animal. Ex vivo-treated cells may optionally be re-introduced into the animal. “Precursor cell”, in the context of the invention, means a living mammalian cell capable of cellular differentiation. Quantitation of a marker can be made using commercially available kits for such a marker. The kits may measure different aspects of such a marker, such as gene expression, protein abundance, protein activity and biochemical or clinical consequences thereof.

“Significantly altering the relative abundance or bioactivity of a marker” means changing the average abundance or biological activity of a protein, mRNA, carbohydrate, lipid, metabolite or other biological analyte whose changed abundance or bioactivity is measurable in a population of cells by practitioners skilled in the art using commercially available kits, wherein the change is shown to occur to a statistically significant degree in cells treated by the methods of this invention compared to a control population. The difference between average measured abundance or bioactivity of a marker is significantly different in a population of cells that received treatment by the methods of this invention (as compared to an untreated control population of cells) if the difference between the two populations is statistically significant, for example, with a calculated probability of p<0.05 using Student's T-test, or other comparable statistical test well known in the art. The abundance or bioactivity of a marker, such as a protein or RNA known to be diagnostic for the differentiated cell population in question, can be readily measured using commercially available test kits, well-known and widely used in the field e.g. ELISA kits, qPCR kits, enzymatic activity kits, etc. Test kits can be purchased for cell surface markers such as CD169, Clec9a, Clec10a, Clec12a, CD205, CD207, CD209, CD209L and MHCII, secreted proteins such as IL-10, TGFbeta, TNFalpha, CCL22 and COL1A1 (collagen), and nuclear proteins such as FoxP3, Nur77/NR4A1, RXRs, PPARs and other modulators of transcrition.

“RXR” means retinoid X receptor, and can refer to either the RXR gene or the protein it specifies. “Rexinoid” means a ligand of an RXR receptor. “RXRs” means any of the RXR isoforms, such as RXR-alpha, RXR-beta, RXR-gamma, and also covers heterodimers formed between them and other nuclear receptors such as NR4As. “NR4As” includes the orphan nuclear receptors NR4A1, NR4A2 and NR4A3, and can refer to either the NR4A gene or the protein it specifies. RXR receptors can and do form functional heterodimers with a variety of other nuclear receptors such as retinoic acid receptors (RARs), thyroid receptors (TRs), vitamin D receptor (VDR), liver X receptors (LXRs), peroxisome proliferator-activated receptors (PPARs), and the aforementioned NR4As.

“CD169” (also known as Siglec-1) means sialoadhesin, a cell adhesion molecule found on the surface of macrophages. Orthologs of this molecule in other mammalian species are included in this definition.

“C-lectins” means a C-type lectin such as Clec4a, Clec9a, Clec10a or Clec12a, and orthologs thereof.

“CCL22” (also known as MDC) means C-C motif chemokine 22, and its orthologs.

“COL1A1” means alpha-1 Type I collagen.

“STING” means “stimulator of interferon genes. STING is also known as TMEM173.

“Nur77” is the protein product of the NR4A1 gene. As an analyte, the two terms are here used interchangeably. Sometimes the gene product may be referred to as Nur77/NR4A1 or NR4A1/Nur77.

This invention provides a method for stimulating levels of differentiation markers in myeloid and non-myeloid mammalian cell lineages in vivo or ex vivo, the method comprising: (i) contacting one or more mammalian cells of a myeloid lineage with a synthetic immodulin peptide about 20 to about 60 amino acids in length and comprising an amino acid sequence corresponding to any one of SEQ ID NOs:1-6; and (ii) contacting one or more mammalian cells of a non-myeloid lineage with said synthetic immodulin peptide; and (iii) measuring a readout of differentiation for the myeloid mammalian cell lineage wherein said readout is accompanied by a significantly increased average level of at least one marker for myeloid lineage differentiation in the contacted cells of the myeloid lineage; and (iv) measuring a readout of differentiation for the non-myeloid mammalian cell lineage wherein said readout is accompanied by a significantly increased average level of at least one marker for non-myeloid lineage differentiation in the contacted cells of the non-myeloid lineage; wherein the at least one marker for myeloid lineage differentiation is CD169, CCL22, Clec4A, Clec9a, Clec10a or Clec12a, and wherein cells of said myeloid and non-myeloid lineages following the contacting steps are co-resident in a living tissue. Under the method, cells of said myeloid and non-myeloid lineages following the contacting steps may come to be co-resident in a living tissue by contacting the synthetic immodulin peptide to each lineage separately or concurrently, wherein “concurrently” shall mean simultaneously, at approximately the same time, or in immediate or overlapping sequence, and having any of the contacting steps take place either in vivo or ex vivo.

Non-myeloid lineages may include cells of a neurogenic lineage, wherein the readout is neurogenesis, and the marker for differentiation is selected from the group consisting of: synaptophysin, synaptopodin, vimentin, NMDAR, AChR, and PSD95. Non-myeloid lineages may also include cells of a myogenic lineage, wherein the readout is myogenesis, and the marker for differentiation is selected from the group consisting of: creatine kinase, myogenin, MYF-4, MYF-5 and actinin-2. Myogenesis includes formation of myotubes as well as higher order structures. Non-myeloid lineages may also include cells of an osteogenic lineage, wherein the readout is osteogenesis, and the marker for differentiation is selected from the group consisting of: alkaline phosphatase, calcified deposits, osteocalcin and BMP-2. Non-myeloid lineages may also include cells of a chondrogenic lineage, wherein the readout is chondrogenesis, and the marker for differentiation is selected from the group consisting of: type II collagen, aggrecan and Sox 9. Non-myeloid lineages may also include cells of a keratinocytic lineage, wherein the readout is differentiated keratinocytes and the marker of differentiation is selected from the group consisting of keratin-10, profilaggrin, loricrin and involucrin. Non-myeloid lineages may also include cells of a lymphocytic lineage, wherein the readout is differentiated lymphocytes and the marker of differentiation is selected from the group consisting of FoxP3 and IL-10. Non-myeloid lineages may also include cells of a pulmonary lineage, wherein the readout is differentiated pulmonary cells and the marker of differentiation is selected from the group consisting of NKX2.1, HOPX, GPRC5A, AGER or AQP5. Non-myeloid lineages may also include cells of a fibroblastic lineage, wherein the readout is differentiated fibroblasts and the marker of differentiation is COL1A1. Non-myeloid lineages may also include cells of a nephrogenic lineage, wherein the readout is differentiated kidney cells and the marker of differentiation is selected from the group consisting of CALB1, AQP2, AVPR2 and PENDRIN. Non-myeloid lineages may also include cells of a hepatogenic lineage, wherein the readout is differentiated liver cells and the marker of differentiation is selected from the group consisting of AAT1, alpha-fetoprotein, HNF-4a, glycogen and albumin.

This invention provides for measuring a readout of differentiation for the non-myeloid mammalian cell lineage. A “readout” may refer to a gene readout (for example, gene transcription), a protein readout (for example by ELISA, or enzymatic assay), a biochemical readout (for example, by assay of a molecule that is not a nucleic acid, peptide or protein) or a functional readout (for example by clinical readout of a patient's symptoms). Transcription can be quantitated in mammalian cells or tissues by techniques such as quantitative PCR (qPCR). Commercially available kits can measure levels of analytes, both protein and non-protein. A clinician's expertise facilitates assessment of a patient's symptoms. For examples, changes in grip strength can be measured with a dynamometer. Body temperature may be measured using a thermometer.

This invention also provides a peptide having an amino terminus formed by covalent linkage to a “small molecule” of molecular mass less than one thousand daltons, preferably less than five hundred daltons. Said “small molecule” is selected from the group consisting of PF-06409577, AICAR, D942, PT1, EX-229, GIT27, GW501516, GW3965, GW9578, RB394, MBX-8025, GW7647, ZLY032, GW590735, GW0742, Amorfrutin B, BMS195614, GW4064, BMS453, FTY720, artesunate, sobetirone, cilofexor, decanoic acid, eicosapentaenoic acid, docosahexanoic acid, lignoceric acid, TTNPB, adapalene, bexarotene, transcinnamic acid, fenofibric acid, ciprofibrate, chlorfibric acid, gemfibrozil, elafibrinor, pioglitazone, roziglitazone, valproic acid, 2-hexyl-4-pentynoic acid, and ibuprofen.

The invention also provides a synthetic immodulin peptide comprising a core sequence selected from the group consisting of any of SEQ ID NOs: 1-6. In some embodiments, said core sequence is aminoterminally extended by a sequence comprising any of sequence ID NOs: 7-14.

In some embodiments the invention provides kinase modulating sequences to be used in conjunction with the immodulin sequences of this invention. Kinase inhibitor peptides have been widely used by practitioners in the field for several decades. For example, U.S. Pat. No. 5,783,405 lists dozens of peptide sequences and teaches their use as protein kinase C inhibitors. Among them are the sequences AFNSYELGS and SLNPEWNET, claimed to inhibit PKC-delta and PKC-beta, respectively, as well as PKC-epsilon stimulating sequences such as NGLLKIK.

In some embodiments the invention provides a synthetic immodulin peptide in a complex with non-covalently bound, purified holotransferrin at about 0.1 to about 10 molar equivalents of one to the other. Holotransferrin is transferrin in which at least one of the iron-binding sites is occupied by ferric iron. The peptide-holotransferrin binary complex provides better targeting of the synthetic immodulin peptide in vivo by virtue of its preferential binding to high-molecular weight size classes of glycosaminoglycans, notably hyaluronan (HMW-HA) which are, in turn, taken up by the receptor CD44 in preferred contexts. The terms “hyaluronan” and “hyaluronic acid” are herein used interchangeably. In some aspects, preferential binding of the binary complex to PIP2, which is enriched in preferred cellular membrane contexts for resolution of inflammation and cellular differentiation, may also (and independently) increase specificity of targeting in vivo.

In some embodiments the invention provides a synthetic immodulin peptide in a complex with non-covalently bound, purified holotransferrin at about 0.1 to about 10 molar equivalents of one to the other, additionally complexed to HMW-HA at about 0.01 to about 100 molar equivalents of purified high-molecular weight hyaluronan relative to peptide. In some aspects, the stability of this ternary complex is stronger at pH 5.2 (endocytic pH) than at pH 7.4, the physiological pH in most cellular compartments. In some aspects, the stability of this ternary complex containing HMW-HA is stronger than the corresponding ternary complex containing LMW-HA. Size classes of hyaluronan are thought to play key roles in inflammation. In some embodiments the invention provides compositions comprising said ternary complex.

In another aspect, the invention provides a method for treating a mammal showing symptoms of immune dysfunction or imbalance comprising administering to said mammal via intramuscular, subcutaneous, parenteral, transdermal, intranasal, intravenous or intrathecal route of administration a pharmaceutical formulation comprising a therapeutically effective dose of an immodulin peptide according to the invention, and a pharmaceutically acceptable excipient, thereby alleviating said symptoms of dysfunction. In some embodiments, the immodulin peptide is administered in a therapeutically effective dose of peptide between about 0.01 mg/kg/day to about 50 mg/kg/day.

In another aspect, the invention provides for co-administration of a helper molecule selected from a group consisting of: ornithine, leucine, raffinose, trehalose, resveratrol, polydatin, ursolic acid, lactate, bile salt, metal, cyclodextrin and cyclic dinucleotide.

This invention envisages an in vitro method for measuring cell differentiating potency of a synthetic immodulin peptide, the method comprising measurement of the abundance of a marker selected from the group consisting of PPARs, RXRs, NR4As, CD169, CCL22, IL-10, TGFbeta, FoxP3, C-lectins, COL1A1, TNFalpha, NfkappaB, MMP-9, IL-6, STING, interferons, RLRs or toll-like receptors in cultured mammalian cells that have been treated with the synthetic peptide. As will be understood by those of skill in the art, the mode of detection of a diagnostic signal will depend on the detection system utilized in the assay. For example, if a fluorescent detection reagent is utilized, the signal may be measured using a fluorometer. If a chemiluminescent detection system is used, then the signal can be detected using a luminometer. Either gene expression (via qPCR) or protein levels and/or enzymatic activity may be measured. Methods for detecting signal from detection systems for such analytes are well known in the art and need not be further described here.

Sequence “identity” and “homology”, as referred to herein, can be determined using BLAST, particularly BLASTp as implemented by the National Center for Biotechnology Information (NCBI), using default parameters. It will be readily apparent to a practitioner skilled in the art that sequences claimed hereunder include all homologous and trivial variants of an immodulin peptide, such as by conservative substitution, extension and deletion in amino acid sequence. Trivial substitution variants include swapping of an amino acid with another belonging to the same class, without such substitution resulting in statistically and functionally significant change. “Classes” of amino acids include positively charged amino acids (arginine, lysine, histidine), negatively charged amino acids (aspartic acid, glutamic acid), aromatic amino acids (tyrosine, phenylalanine, tryptophan), branched chain amino acids (valine, leucine isoleucine) and other natural groupings such as (serine, threonine) and (asparagine, glutamine). For the purposes of this invention, such conservative substitutions to immodulin sequences, if they do not create a significant change in function, are considered equivalent to the original and are covered by the scope of this invention.

For testing efficacy of pharmaceutical composition containing an immodulin peptide, an effective amount of therapeutic agent is administered to a subject having a disease. In some embodiments, the agent is administered at about to about 50 milligrams per kilogram total body weight per day (mg/kg/day). In some embodiments the agent is administered at about 0.001 to about 50 mg/kg/day, e.g., 0.01, 0.015, 0.02, 0.05, 0.1, 0.2, 0.5, 0.7, 1, 2, 4, 5, 7, 9, 10, 15, 25, 30, 35 or another dose from about 0.001 mg/kg/day to about 50 mg/kg/day.

Therapeutic agents are preferably administered via oral or parenteral administration, including but not limited to intravenous (IV), intra-arterial (IA), intraperitoneal (IP), intramuscular (IM), intracardial, subcutaneous (SC), intrathoracic, intraspinal, intradermal (ID), transdermal, oral, sublingual, inhaled, and intranasal routes. IV, IP, IM, and ID administration may be by bolus or infusion administration. For SC administration, administration may be by bolus, infusion, or by implantable device, such as an implantable minipump (e.g., osmotic or mechanical minipump) or slow release implant. The agent may also be delivered in a slow release formulation adapted for IV, IP, IM, ID or SC administration. Inhaled agent is preferably delivered in discrete doses (e.g., via a metered dose inhaler adapted for protein delivery). Administration of a molecule comprising an agent via the transdermal route may be continuous or pulsatile. Administration of agents may also occur orally. For parenteral administration, compositions comprising a therapeutic agent may be in dry powder, semi-solid or liquid formulations. For parenteral administration by routes other than inhalation, the composition comprising an agent is preferably administered in a liquid formulation. Compositions comprising an agent formulation may contain additional components such as salts, buffers, bulking agents, osmolytes, antioxidants, detergents, surfactants, and other pharmaceutical excipients as are known in the art.

As will be understood by practitioners skilled in the art, the symptoms of disease alleviated by the instant methods, as well as the methods used to measure the symptom(s) will vary, depending on the particular disease and the individual patient. All references cited in this document, including patent applications and publications cited therein, are incorporated by reference in their entirety.

EXAMPLES Example 1. Binding of Phosphatidylinositol Phosphates (PIPs) by Immodulin Peptides

N-terminally biotinylated versions of the peptides listed below were used in various assays listed in the examples. For the two types of phosphatidylinositon phosphate (PIP)-binding assays, imm1-imm6 peptides were used.

PEPTIDE SEQUENCE None (buffer) imm1 KNGFYHSRQCETSMDGEAGLCW imm2 KHGLYNLKQCKMSLNGQRGECW imm3 KKGFYKKKQCRPSKGRKRGFCW imm4 RNGNFHPKQCHPALDGQRGKCW imm5 RKGFYKRKQCKPSRGRKRGICW imm6 HRGFYRKRQCRSSQGQRRGPCW immX3AVD KKGFYKKKQCRPSKGRKRGFCWAVD immX4AVD RNGNFHPKQCHPALDGQRGKCWAVD immX5AVD RKGFYKRKQCKPSRGRKRGICWAVD immX3dAVD KKGFYKKKQCRPSKGRKRGFCW(dA)VD immX3dAdVdD KKGFYKKKQCRPSKGRKRGFCW(dA)(dV)(dD) immX3dSdVdD KKGFYKKKQCRPSKGRKRGFCW(dS)(dV)(dD) immX3K4e+1 NGLLKIKKGFYKKKQCRPSKGRKRGFCWAVD immX3K4e+2 HDAPIGYDKKGFYKKKQCRPSKGRKRGFCWAVD immX3K4edSdVdD NGLLKIKKGFYKKKQCRPSKGRKRGFCW(dS)(dV)(dD) immX3K1dAdVdD SLNPEWNETKGFYKKKQCRPSKGRKRGFCW(dA)(dV)(dD) Underlined residues are D-amino acids

In the PIP-binding membrane assay, 400 ng of each peptide was bound to Membrane Lipid Strips (Catalog # P-6002, Echelon Biosciences, Salt Lake City, UT) and developed according to the manufacturer's instructions. Binding to PIPs was as follows (−−− no binding; +++ strong binding):

PtdIns(4)P PtdIns(4,5)P PtdIns(3,4,5)P PEPTIDE Phosphatidylinositol [PIP1] [PIP2] [PIP3] imm1 — — — — imm2 — — — — imm3 — — +++ ++ imm4 — — — — imm5 — ++ +++ ++ imm6 — — + —

In the plate assay, biotinylated peptides imm3 and imm5 were bound to 96-well, streptavidin-coated plates (BioLegend corporaton, San Diego, CA) at 400 ng per well in PBS buffer at room temperature for 60 mins. Plates were washed in PBS and developed with FITC-labelled PIP2 (purchased from Cayman Chemical Co, Ann Arbor, MI). Fluorescence was counted in a laboratory fluorescence spectrophotometer. The results were as follows (EDTA background set to =100; **p<0.01 vs background control):

Background PEPTIDE Control Zinc Ferric Iron imm3 1.0 ± 0.8 4.7 ± 1.3** 26.2 ± 5.7** imm5 1.8 ± 0.5 5.0 ± 1.2** 23.4 ± 3.5**

Example 2. Enhanced Myeloid Cell Differentiation Stimulated by Immodulin Peptides

Mammalian cell differentiation assays employed the THP1-Dual monocyte reporter cell line (Invivogen Inc, San Diego, CA) seeded at 2×10e5 cells per well in 96-well plates and cultured at 37 degrees C. in RPMI-1640 growth medium plus 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were subsequently treated for 24 hours with 100 ng/ml Phorbol 12-myristate 13-acetate (PMA protocol; Cayman Chemical Company, Ann Arbor, MI). Peptide (0.33 uM or 0.66 uM, as indicated) was then added, and incubation continued for an additional 24 hours. Culture supernatants were assayed for CCL22. Plates with adherent cells were washed with PBS and assayed for immunoreactivity of surface markers such as CD169, Clec9a or Clec12a using fuorescent tag- or biotin-labeled anti-human antibodies purchased from Miltenyi Biotec (Auburn, CA) and the secondary detection reagent recommended by the manufacturer. Results were expressed as arbitrary units relative to the control immX3AVD peptide, and p values were also calculated and shown relative to the control immX3AVD peptide (values significantly above background are shown in bold font; **p<0.05 versus immX3AVD control; AU=arbitrary immunoreactivity units relative to control, avg±SD).

PEPTIDE CD169+ (AU) CCL22 pg/ml Clec12A(AU) None (buffer) 1.2 ± 1.1**  17.1 ± 1.2**   0.5 ± 3.2** imm1 3.1 ± 2.2** n.d. n.d. imm2 3.0 ± 0.5** n.d. n.d. imm3 27.8 ± 3.0**    55.3 ± 16.1** n.d. imm4 3.8 ± 1.1** n.d. n.d. imm5 36.5 ± 3.0**  n.d. n.d. imm6 19.9 ± 2.3**  n.d. n.d. immX3AVD 100 282.4 ± 18.4  100 immX4AVD 1.0 ± 0.3** n.d. n.d. immX5AVD 100.9 ± 3.5     228.6 ± 8.1   n.d. immX3dAVD 104.3 ± 6.1     247.3 ± 16.1  n.d. immX3dAdVdD 183.3 ± 6.4**   n.d. 106.6 ± 5.2   immX3dSdVdD 145.7 ± 9.3**   n.d. n.d. immX3FVS 109.4 ± 4.5     251.3 ± 5.8   n.d. immX3RVD 88.1 ± 4.2    272.5 ± 35.7  n.d. immX3K4e + 1 128.4 ± 1.4**   n.d n.d immX3K1dAdVdD 249.3 ± 13.9**   135.8 ± 24.2**  321.9 ± 25.6** AU: arbitrary units (immunoreactivity); #: PMA protocol; ## cytokine protocol; n.d. = not determined

Example 3. Adjuvant Effect of Molecules on Immodulin Peptide Potency in THP1-Dual Assay

The THP1-Dual cell differentiation assay was carried out as described above in Example 2, using immX3 peptide (330 nM) in all samples plus the indicated helper or inhibitor molecule. Molecules were purched from Cayman Chemical Company (Ann Arbor, MI) or Sigma Chemical Company (St. Louis, MO). AU=arbitrary ELISA units. **p<0.01.

HELPER CAS# CLASS [CONC] CD169 (AU) None (buffer) 100.0 ± 4.9   Holotransferrin (Tf) Fe-binding protein 0.8 ug/ml 126.8 ± 9.3** Hyaluronan(HMW-HA) glycosaminoglycan 0.6 ug/ml 110.5 ± 10.2  HMW-HA + Tf 2.0 ug/ml #  82.1 ± 7.5** LMW-HA + Tf 2.0 ug/ml # 120.6 ± 7.8   Heparin glycosaminoglycan 1.0 ug/ml  34.1 ± 2.9** Supercinnamaldehyde 70351-51-8 C/EBPb inhibitor 2 uM  159.7 ± 16.6** Bisindolylamide 138489-18-6 PKC inhibitor 2 uM 130.1 ± 8.4** RIG-I agonist [a] RIG-I agonist 1 ug/ml 135.4 ± 9.1** G10 702662-50-8 STING agonist 2 uM  143.4 ± 17.8** 2′3′-cGAMP Cyclic dinucleotide 15 uM  168.5 ± 10.9** Cyclic-di-GMP Cyclic dinucleotide 15 uM 142.9 ± 9.7** D-mannoheptulose Sugar 50 uM  140.8 ± 12.4** D-raffinose Sugar 1 mM 111.9 ± 4.0** L-lactate Acid 300 uM 121.1 ± 5.6** L-fumarate Acid 1 mM 101.5 ± 7.3   HP-B-cyclodextrin Starch 1 mM 121.1 ± 4.6** Calcitriol 32222-06-3 VDR agonist 2 uM 102.0 ± 11.3  Spironolactone 52-01-7 RXR or NR4A ligand 2 uM  120.1 ± 10.5** C-DIM12 178946-89-9 RXR or NR4A ligand 2 uM 119.0 ± 3.7** C-DIM8 151358-47-3 RXR or NR4A ligand 2 uM 91.0 ± 24.8 Clobetasol 25122-46-7 RXR or NR4A ligand 2 uM 98.5 ± 5.5  Cilostazol 73963-72-1 RXR or NR4A ligand 2 uM 99.4 ± 27.7 Cytosporone B 321661-62-5 RXR or NR4A ligand 2 uM 120.3 ± 6.4** Dihydroergotamine 6190-39-2 RXR or NR4A ligand 2 uM  170.3 ± 20.7** 6-mercaptopurine 6112-76-1 RXR or NR4A ligand 2 uM 104.9 ± 4.0   Bexarotene 153559-49-0 RXR or NR4A ligand 2 uM 96.9 ± 6.8  LG100268 153559-76-3 RXR or NR4A ligand 2 uM   76.8 ± 11.9** HX600 172705-89-4 RXR or NR4A ligand 2 uM 110.5 ± 19.0  HX531 188844-34-0 RXR or NR4A ligand 2 uM 90.4 ± 6.2  Cyclosporine A NFAT inhibitor 2 uM 128.9 ± 6.7** GM-CSF Growth factor class 0.1 ug/ml  147.0 ± 15.3** CSF1 Growth factor class 0.1 ug/ml 108.0 ± 32.3  Leptin Growth factor class 1.0 ug/ml 103.3 ± 11.6  GIT27 6501-72-0 TLR4 inhibitor 2 uM  166.5 ± 34.1** [a] Invivogen Inc. (San Diego, CA) Cat.# tlrl-3prnalv; # each helper at 2 ug/ml;

Example 4. Binding to Immodulin Peptides to Transferrin and Glycosaminoglycans

Effects of pH, PIP2 and size classes: Streptavidin-coated 96-well plates (G-Biosciences, St. Louis, MO) were pre-loaded with biotinylated peptides [see Example 1] at 400 ng/well, at room temperature for 60 minutes. The plate was washed with PBS buffer and then 1 ug FITC-labelled heparin or hyaluronic acid (FITC-HMW-HA) in 100 uL PBS buffer was added per well (all tests done in quadruplicate). Incubation proceeded for 90 minutes at room temperature, followed by two PBS washes. The plate was read in a standard laboratory fluorometer (488/525 nm exitation/absorbance) and the counts normalized for immX3 peptide binding=100. Values statistically above background are shown in bold font. **p<0.05 versus immX3AVD.

PEPTIDE Heparin Hyaluronate imm2 27.4 ± 22.9 3.1 ± 9.8 imm3 104.2 ± 22.0   57.2 ± 5.7** imm4  6.8 ± 24.1 14.6 ± 22.6 imm5 95.1 ± 25.7  60.0 ± 8.6** imm6 97.2 ± 17.3   44.9 ± 14.1** immX3AVD 100.0 ± 4.7   100.0 ± 22.8  immX4AVD  7.3 ± 17.1 61.1 ± 34.1 immX5AVD 68.7 ± 26.2 113.9 ± 15.7  immX3dAdVdD  55.5 ± 5.7** 104.2 ± 14.7  immX3K1dAdVdD  56.0 ± 7.3** 101.9 ± 16.4 

A second binding experiment was done as above, but using either 6 mM sodium phosphate buffer pH 7.4 or sodium citrate buffer pH 5.2 in the presence of FITC-HMW-HA (1.5 ug/well), holotransferrin (10 ug/well) and excess PIP2 (500 ng/well), LMW-HA (hexamer; 10 ug/well) or HMW-HA (10 ug/well). **p<0.05 versus peptide binding control (=100);

pH Peptide CONTROL +cold PIP2 +cold LMW-HA +cold HMW-HA 7.4 None (buffer)   4.3 ± 3.8** n.d. n.d. n.d. 7.4 immX3AVD 100.0 ± 8.3    10.4 ± 8.2**   92.6 ± 11.8** 18.5 ± 3.7** 7.4 immX5AVD 100.0 ± 7.6    53.7 ± 2.1** 94.9 ± 11.4 22.6 ± 8.8** 5.2 None (buffer)  27.3 ± 8.4** n.d. n.d. n.d 5.2 immX3AVD  140.0 ± 16.7** 92.2 ± 29.4 100.6 ± 15.9  12.5 ± 7.1** 5.2 immX5AVD  66.6 ± 7.2**  52.8 ± 4.0** 76.8 ± 5.9   9.6 ± 6.5** n.d. = not determined;

A third binding experiment was done as described above using immX3AVD peptide with or without 10 ug/well holotransferrin (holoTf) plus either 10 ug/well LMW-HA or either 1.5 ug or 10 ug HMW-HA/well in the binding step, followed by a buffer wash step. 100 ng/well pure recombinant CD44 protein (Abcam, Waltham, MA) was then added to the indicated wells, incubated for 60 minutes at room temperature and the plate was washed with buffer. Anti-CD44 monoclonal antibody (Abcam, Waltham, MA) was added towells at 1:200, incubated for 60 minutes at room temperature and the plate was then washed and developed by secondary antibody and TMB detection as recommended by the manufacturer. CD44 immunoreactivity was expressed in arbitrary immunoreactivity units (AU), with **p<0.05 versus immx3AVD+1.5 ug HMW-HA control, which was set to=100 AU. Results are shown below:

PH Peptide CONTROL 1.5 ug/w HMW-HA 10 ug/w HMW-HA 10 ug/w LMW-HA 7.4 None (buffer)   1.5 ± 15.8** n.d. n.d. n.d. 7.4 immX3AVD  19.6 ± 13.0** 100.0 ± 15.5  101.1 ± 4.9   103.0 ± 19.4  7.4 immX3AVD +   3.1 ± 11.5** 98.3 ± 11.0 107.0 ± 1.9    38.7 ± 4.4** holoTf 5.2 None (buffer)   0.9 ± 21.5** n.d. n.d. n.d. 5.2 immX3AVD  13.2 ± 22.0** 100.0 ± 4.6   105.1 ± 11.4  102.6 ± 17.9  5.2 immX3AVD +  12.0 ± 21.2** 103.3 ± 15.7  94.8 ± 9.5   27.4 ± 6.5** holoTf n.d. = not determined;

Example 5. Non-myeloid Cell Differentiation Stimulated by Immodulin Peptides and Compounds

Differentiation assays employed the C2C12 myoblastic cell line (ATCC, Manassas, VA) seeded at 2×10e5 cells per well in 96-well plates and cultured at 37 degrees C. in DMEM growth medium plus 10% fetal bovine serum and 1% penicillin/streptomycin. For differentiation, 10% fetal bovine serum was replaced with 2% horse serum. 0.66 uM peptide was then added, and incubation continued for an additional 96 hours. Plates with adherent cells were washed with PBS, lysed with Lytic-M buffer (Sigma Chemical Company, St. Louis, MO) and the lysates were assayed for creatine kinase using a kit purchased from Bioassay Systems (Cat. #ECPK-100, Hayward, CA). Effects were visually confirmed with microscopy tracking myotube formation. Results were expressed as units per milliliter relative to the control immX3AVD peptide, and p values were also calculated and shown relative to control immX3AVD peptide (** p<0.05 versus immX3AVD control; avg±SD).

PEPTIDE Creatine Kinase Act. Helper molecule None (buffer)   0.7 ± 2.6** none immX3AVD 100.0 ± 2.2   none immX3AVD 120.6 ± 2.5** Tf immX3AVD 136.7 ± 13.3  Tf + LMW-HA (2 ug/ml) immX3AVD  33.2 ± 3.2 # Tf + HMW-HA (2 ug/ml) immX3AVD 161.5 ± 6.5** L-ornithine (1 mM) immX3AVD 140.3 ± 8.3** D-raffinose (1 mM) immX3AVD 174.2 ± 5.0** GW 501516 (2 uM) immX3AVD  67.4 ± 5.2** GW 3965 (2 uM) immX3AVD 109.4 ± 3.2** ciprofibrate (2 uM) immX3dAdVdD 132.8 ± 5.2** none immX3dSdVdD  155.6 ± 14.1** none immX3K4e + 1 178.4 ± 7.6** none # p < 0.05 vs immX3AVD + Tf; Tf = holotransferrin at 2 ug/ml; L/HMW-HA = low/high-molecular wt hyaluronan; Another non-myeloid lineage was also tested. Primary human T-cells pre-selected for recognizing GAD65 peptide antigen, and the peptide antigen itself were purchased from Cellero LLC (Memphis, TN). THP1-Dual cells were treated with both GAD65 peptide antigen (1 ug/ml) and immodulin peptide (0.66 uM) using the protocol described in Example 2. After completion of the myeloid cell differentiation step (24 hours), supernatants were collected and 100,000 primary T-cells were added to each well in RPMI-1640 medium (100 uL per well). Plates were incubated for an additional 96 hours and cell extracts were prepared using CellLytic-M reagent (Sigma Chemical Co., St Louis, MO). Supernatants were assayed for CCL22, IL-10 and TGFbeta, and cell extracts assayed for FoxP3, all by ELISA using reagents from R&D Systems, Minneapolis, MN. The results are shown in the table below (**p<0.05 vs immX3AVD, set to=100):

PEPTIDE GAD65 CCL22 (pg/ml) IL-10 (pg/ml) TGFb (pg/ml) FoxP3 (AU) None None   5.8 ± 1.3**    160 ± 86.1**  15.4 ± 0.4**   23.4 ± 10.1** immX5AVD 1 ug/ml  58.7 ± 2.7**   870 ± 8.2**  52.8 ± 7.4**  14.8 ± 4.3** immX3AVD 1 ug/ml 88.8 ± 13.6  1331 ± 94.3  131.4 ± 41.4  100.0 ± 20.5  immX3dSdVdD 1 ug/ml 77.8 ± 2.0   1534 ± 176.4 87.6 ± 10.7 176.6 ± 8.0** immX3K1dSdVdD 1 ug/ml  554.5 ± 59.3**    7038 ± 168.1**  780.7 ± 51.7** 205.5 ± 8.4** Yet another non-myeloid lineage was tested. Osteoblast cell line MC3T3E1 was purchased from ATCC (Manassas, VA) and grown in 96-well plates in alpha-MEM medium supplemented with 50 ug/ml ascorbic acid and 5 mM beta-glycerophosphate. Peptides were added and incubation proceeded for 5 days. Cells were washed with PBS buffer and extracts prepared using 100 uL 1X RIPA buffer (Cayman Chemical Company; Ann Arbor, MI) per well. Alkaline phosphatase was measured using 0.37 mg/ml 4-nitrophenyl phosphate in DEA buffer (both reagents purchased from Cayman Chemical Company) by measuring the slope of absorbance at 405 nm using a spectrophotometer. 10 uL of RIPA extract was used for each assay. Separately, protein concentration of extract was measured using a commercial kit. Values were corrected for protein concentration. Results are shown below (**p<0.05 vs immX3AVD=100):

PEPTIDE (0.66 uM) Compound added Alk. Phos. Activity immX3AVD None 100.0 ± 0.5   immX3AVD 1 mM L-ornithine 159.7 ± 4.4** immX3AVD 1 mM D-raffinose 134.6 ± 4.6** immX3AVD 2 uM Ciprofibrate 127.2 ± 2.7** immX3AVD 2 uM GW 501516 168.7 ± 3.0** immX3AVD 2 uM GW 3965  59.1 ± 4.3**

Example 6. N-terminal Modification of Peptide with Small Molecules of Molecular Mass below One Thousand Daltons that are not Amino Acids

Similar results were obtained for covalent attachment of small molecules to an immodulin peptide or to a D-tetrapeptide dLys-dAsp-dLys-dPro, with similar efficiencies of coupling to either peptide, thereby demonstrating the generality of the method. Peptides were synthesized according to a common Fmoc/tBu solid phase synthesis strategy well-known in the art. Synthesis may be manual of automated. After the peptide synthesis the resin was divided into batches of 20 umol. Each batch was treated with one of the organic compounds specified in the table shown immediately below. The coupling was carried out using 2 equivalents of the compound, 2.4 equivalents of activator HATU or HCTU, and 4 equivalents of NMM base. The reaction mixture was renewed after 2 hrs reaction time and allowed to react another 4 hrs or overnight. After washing the resin several times with DMF, and subsequently with DCM, the batches were dried. For the cleavage of the peptides from the resin the resins were treated with 1% DTT, 2% water and 3% TIPS in TFA for 3.5 hrs. The cleavage solution was separated from the resin and treated with diethylether/n-pentane (1:1). The resulting precipitate was centrifuged and the pellet washed three times in the same DEE/pentane mixture. The recovered peptide was air dried and stored at −20 degrees C. or further purified by HPLC using a 0-50% acetonitrile gradient, 0.1% trifuoroacetic acid (20 min). The results of the above conjugation experiments show that, both inter-class and intra-class, there is wide variation in conjugation efficiency from compound to compound. It appears that the chance of practical success (>80% correct yield, for instance) for each instantiation of this technology is less than 50% until tested.

Class Compound CAS No. MW Yld T4* Yld IM3* fatty acid oleic acid 112-80-1 282.5 44.21% fatty acid eicosapentaenoic acid 10417-94-4 302.5 66.79% fatty acid lignoceric acid 557-59-5 368.6 89.20% fatty acid decanoic acid 1002-62-6 172.2 88.67% 98.0% fatty acid docosahexanoic acid 6217-54-5 368.6 57.77% fatty acid lauric acid 143-07-7 200.3 85.14% 96.7% fatty acid 10-hydroxy-2-decenoic acid 14113-05-4 186.3 44.38% phenolic acid ferulic acid 1135-24-6 194.2 26.58% phenolic acid isoferulic acid 537-73-5 194.2 55.80% 70.2% phenolic acid Aspirin 50-78-2 180.2 56.5% phenolic acid valeroyl salicylate 64206-54-8 222.2 11.76% pentacyclic betulinic acid 472-15-1 456.7    <1% anthraquinone Rhein 478-43-3 284.2 50.95% anthraquinone Diacerein 13939-02-1 368.3  43.2% xanthone 2,7-dichlorodihydro- 4091-99-0 487.3  91.2% proprionic acid (s)-ketoprofen 22161-81-5 254.3 77.86% proprionic acid Ibuprofen 15687-27-1 206.3 93.42% 98.0% carboxylic acid trans-cinnamic acid 140-10-3 148.2 93.12% 81.5% carboxylic acid (s)-(−)-perillic acid 23635-14-5 166.2 27.96% carboxylic acid fenofibric acid 42017-89-0 318.8 85.67% 99.9% indoleacetic acid Indomethacin 53-86-1 357.8  87.5%  85.2%# pentanoic acid valproic acid 1069-66-5 144.2 91.43% 84.9% alkynoic acid 2-hexyl-pentynoic acid 96017-59-3 182.3 85.1% indolylcarboxylic RG-108 48208-26-0 334.3   74.3%@ retinoid all-trans retinoic acid 302-79-4 300.4  13.1% rexinoid Bexarotene 153559-49-0 348.5 97.09% 94.4% *% yield by MS for T4 (tetrapeptide) and imm3 peptide (>80% in bold type); #lost p-chlorophenone group; @indole core oxidized by Arg (protecting gp);

Example 7. Collagen Stimulating Activity of Modified Immodulin Peptides

Immodulin peptides have potential untility in the field of cosmetics. HFF-1 human fibroblast cell line was obtained from the American Type Culture Collection (ATCC). Cells were grown in a T-75 flask in DMEM Medium containing 10% fetal bovine serum and penicillin-streptomycin at 37° C. in a humidified, 5% CO₂ incubator. Cells (100 ml, 2,000 cells/well) were plated in a 96-well plate and incubated overnight at 37° C. in a humidified, 5% CO₂ incubator. Next day, 10 ml/well of compounds were added (quadruplicate wells). After 72 hour incubation with the compound, supernatants were collected for Collagen-1 ELISA assay and cell viability was measured in a luminometer after the addition of 100 mL/well CellTiterGlo reagent (Promega Inc, Madison, WI) as recommended by the manufacturer. Collagen stimulating activity of immodulin peptides in HFF-1 dermal fibroblasts: Peptides were added to cells at 2 uM. Collagen-1A1 (COL1) immunoreactivity was measured in the supernatants of cultured cells by ELISA using a rabbit monoclonal anti-COL1 primary antibody (Abcam, Cambridge, MA). The results of this experiment are shown in the Table below. Control (buffer) value of immunoreactivity was set to 100. The data show that collagen stimulating activity of various immodulin peptides N-modified with small molecules are influenced by the specific amino acid extension sequences and by the N-terminally conjugated carboxylic acids. *p<0.05, **p<0.01 vs “no peptide” control; bex=bexarotene; isf=isoferulic; vlp=valproic; dec=decanoic; cin=cinnamic; rhn=rhein;

Peptide Sequence COL1 No peptide 100 imm3 KKGFYKKKQCRPSKGRKRGFCW 101 imm4 RNGNFHPKQCHPALDGQRGKCW 105 imm5 RKGFYKRKQCKPSRGRKRGICW  93 imm3bex (bex)-KKGFYKKKQCRPSKGRKRGFCW 102 imm3isf (isf)-KKGFYKKKQCRPSKGRKRGFCW 131* imm3vlp (vlp)-KKGFYKKKQCRPSKGRKRGFCW 157** imm3dec (dec)-KKGFYKKKQCRPSKGRKRGFCW 114 imm3cin (cin)-KKGFYKKKQCRPSKGRKRGFCW 106 imm3rhn (rhn)-KKGFYKKKQCRPSKGRKRGFCW  68** imm3K9 AFNSYELGSKGFYKKKQCRPSKGRKRGFCW 155** imm3K9.1 AFNSYELGSKKGFYKKKQCRPSKGRKRGFCW 156** imm3K9c AFNSYELGSKGFYKKKQCRPSKGRKRGFCWAVDKY 158** imm3K8 FNSYELGSLKKGFYKKKQCRPSKGRKRGFCW  98

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

What is claimed is:
 1. A method for stimulating levels of differentiation markers in myeloid and non-myeloid mammalian cell lineages in vivo or ex vivo, the method comprising: (i) contacting one or more mammalian cells of a myeloid lineage with a synthetic immodulin peptide about 20 to about 60 amino acids in length and comprising an amino acid sequence corresponding to any one of SEQ ID NOs:1-6; and (ii) contacting one or more mammalian cells of a non-myeloid lineage with said synthetic immodulin peptide; (iii) measuring a readout of differentiation for the myeloid mammalian cell lineage wherein said readout is accompanied by a significantly increased average level of at least one marker for myeloid lineage differentiation in the contacted cells of the myeloid lineage and wherein the at least one marker for myeloid lineage differentiation comprises CD169; and (iv) measuring a readout of differentiation for the non-myeloid mammalian cell lineage wherein said readout is accompanied by a significantly increased average level of at least one marker for non-myeloid lineage differentiation in the contacted cells of the non-myeloid lineage; wherein cells of said myeloid and non-myeloid lineages following the contacting steps are co-resident in a living tissue.
 2. The method according to claim 1, wherein the at least one marker for myeloid differentiation comprises a marker selected from the group consisting of: CCL22, Clec4A, Clec9a, Clec10a and Clec12a.
 3. The method according to claim 1, wherein the one or more mammalian cells of a non-myeloid lineage are from a neurogenic lineage, the readout is neurogenesis, and the at least one marker for non-myeloid differentiation is selected from the group consisting of: synaptophysin, synaptopodin, vimentin, NMDAR, AChR, and PSD95.
 4. The method according to claim 1, wherein the one or more mammalian cells of a non-myeloid lineage are from a myogenic lineage, the readout is myogenesis, and the at least one marker for non-myeloid differentiation is selected from the group consisting of: creatine kinase, myogenin, MYF-5 and actinin-2.
 5. The method according to claim 1, wherein the one or more mammalian cells of a non-myeloid lineage are from a osteogenic lineage, the readout is osteogenesis, and the at least one marker for non-myeloid differentiation is selected from the group consisting of alkaline phosphatase, calcified deposits, osteocalcin and BMP-2.
 6. The method according to claim 1, wherein the one or more mammalian cells of a non-myeloid lineage are from a fibroblastic lineage, the readout is dermal regeneration, and the at least one marker for non-myeloid differentiation is Col1A1.
 7. The method according to claim 1, wherein the one or more mammalian cells of a non-myeloid lineage are from a lymphocytic lineage, the readout is regulatory lymphocyte proliferation, and the at least one marker for non-myeloid differentiation is selected from the group consisting of: FoxP3 and IL-10.
 8. The method according to claim 1, wherein the synthetic immodulin peptide is covalently linked to at least one small non-amino acid molecule of molecular mass less than one thousand daltons.
 9. The method according to claim 8, wherein the small non-amino-acid molecule is selected from a group consisting of: PF-06409577, AICAR, D942, PT1, EX-229, GIT27, GW501516, GW3965, GW9578, RB394, MBX-8025, GW7647, ZLY032, GW590735, GW0742, Amorfrutin B, BMS195614, GW4064, BMS453, FTY720, artesunate, sobetirone, cilofexor, decanoic acid, eicosapentaenoic acid, docosahexanoic acid, lignoceric acid, TTNPB, adapalene, bexarotene, transcinnamic acid, fenofibric acid, ciprofibrate, chlorfibric acid, gemfibrozil, elafibrinor, pioglitazone, roziglitazone, valproic acid, 2-hexyl-4-pentynoic acid, and ibuprofen.
 10. The method according to claim 8, wherein the synthetic immodulin peptide is co-administered with a helper molecule selected from a group consisting of: ornithine, leucine, raffinose, trehalose, resveratrol, polydatin, ursolic acid, lactate, bile salt, metal, cyclodextrin and cyclic dinucleotide.
 11. The method according to claim 8, wherein the amino acid sequence of said synthetic immodulin peptide further comprises any of SEQ ID NOs: 7-14.
 12. The method according to claim 8, wherein the synthetic peptide is provided in a complex with non-covalently bound, purified holotransferrin at about 0.1 to about 10 molar equivalents.
 13. The method according to claim 12, wherein said synthetic peptide-holotransferrin complex further comprises about 0.01 to about 100 molar equivalents of purified high-molecular weight hyaluronan, wherein binding of said synthetic immodulin peptide-holotransferrin complex to high-molecular weight hyaluronan is significantly stronger at pH 5.2 than at pH 7.4.
 14. The method according to claim 12, wherein said synthetic peptide-holotransferrin complex further comprises about 0.01 to about 100 molar equivalents of purified high-molecular weight hyaluronan, wherein binding of said synthetic immodulin peptide-holotransferrin complex to high-molecular weight hyaluronan is significantly stronger than binding of said synthetic immodulin peptide-holotransferrin complex to low-molecular weight hyaluronan at pH 7.4.
 15. (canceled)
 16. A composition comprising a pH-sensitive non-covalent complex of: (i) a synthetic immodulin peptide about 20 to about 60 amino acids in length; (ii) about 0.1 to about 10 molar equivalents of purified holotransferrin; and (iii) about 0.01 to about 100 molar equivalents of purified high-molecular weight hyaluronan, wherein binding of said high-molecular weight hyaluronan within said pH-sensitive non-covalent complex is significantly stronger at pH than at pH 7.4.
 17. A composition comprising a size-sensitive non-covalent complex of: (i) a synthetic immodulin peptide about 20 to about 60 amino acids in length; (ii) about 0.1 to about 10 molar equivalents of purified holotransferrin; and (iii) about 0.01 to about 100 molar equivalents of purified high-molecular weight hyaluronan, wherein binding of said high-molecular weight hyaluronan within said size-sensitive non-covalent complex is significantly stronger than binding to low-molecular weight hyaluronan at pH 7.4. 